It has been shown that the enzyme-inhibitor described by Bowman, Proc. Soc. Exptl. Med. 1946, 63, 547, and Birk et al., Bull. Res. Council Israel 1962, Sec. A 11, 48 and Biochim. Biophys. Acta 1963, 67, 326, and subsequently referred to as the Bowman-Birk Inhibitor (BBI), possesses certain physiological activity that prevents, or at least greatly reduces, radiologically or chemically induced malignant transformation of cells in culture and in experimental animals.
Yavelow et al., Proc. Natl. Acad. Sci. USA 1985, 82, 5395-5399, reported that a crude soybean extract, if defatted with acetone, effectively blocked cell transformation in vitro. An active component of this crude extract is the BBI. These observations, with epidemiological data, suggested BBI as a putative dietary anticarcinogen, particularly with respect to colon cancer.
Weed et al., Carcinogenesis 1985, 6,1239-1241, disclose that an extract of soybeans containing the Bowman-Birk protease inhibitor added to the diet of dimethylhydrazine (DMH)-treated mice resulted in a significant suppression of odenomatous tumors of the colonic mucosa. Weed et al. noted that DMH-induced colon cancer in mice is generally regarded as an excellent animal model for the human disease, with carcinogen treatment inducing adenocarcinomas of the colon and rectum which are similar to the tumors arising in the human colon. The Weed et al. study suggested the possibility that a dietary additive of the sort studied might confer some protection against the development of human colon cancer without undesirable side effects. The BBI extract and methods for its preparation were as described by Yavelow et al., Proc. Natl. Acad. Sci. USA 1985, 82, 5395-5399.
Messadi et al., JNCI 1986, 76,447-452, demonstrated that a soybean extract containing the protease inhibitor BBI suppresses 7,12-dimethyl-benz[a]anthracene (DMBA)-induced carcinogenesis in the hamster cheek pouch. This oral cancer model, with the use of the hamster check pouch carcinogenesis system, has the same histopathology, growth pattern, and precancerous lesions as the most common form of human oral cancer, squamous cell carcinoma. It was shown in this study that hamster cheek pouch carcinogenesis can be inhibited by BBI and suggested that human oral carcinogenesis might respond to BBI in a comparable manner. The BBI preparation used in this study was a crude extract of the inhibitor prepared as described by Yavelow et al., supra.
Baturay et al., Cell Biology and Toxicology 1986, 2, 21-32, disclose that a BBI preparation, wherein a crude soybean extract is defatted with acetone, suppresses radiation and chemically induced transformation in vitro, with or without enhancement by the co-carcinogen, pyrene. Yavelow et al., supra, show that either pure BBI or the BBI extract prepared in accordance with their methods suppresses radiation induced transformation in C3H10T1/2 cells. Kennedy et al., Proc. Nat'l. Acad. Sci. USA 1984, 81, 1827-39, report that either pure BBI or the BBI extract prepared in accordance with their method reduce the levels of chromosome abnormalities in cells of patients with Bloom's syndrome (a genetic disease in which the high levels of chromosome abnormalities are thought to predispose the patients to a higher than normal cancer incidence). Still, other studies suggest that soybean-derived protease inhibitors can have suppressive effects on skin, breast and liver carcinogenesis in vivo.
Kennedy et al., Anticarcinogenesis and Radiation Protection, Cerutti et al., Eds., Plenum Pub, 1987, pp. 285-295, disclose that BBI suppresses carcinogenesis in various systems using a crude BBI extract prepared by defatting soybeans with acetone. Their results suggested that very low concentrations of BBI-type protease inhibitor preparations would be effective as chemopreventative agents for colon cancer. There was no evidence to suggest that the use of protease inhibitors as chemopreventative agents would be complicated by possible toxicity problems.
St. Clair et al., Cancer Res. 1990, 50,580-586 report that the addition of 0.5% or 0.1% semi-purified BBI or 0.1% or 0.01% purified BBI to the diet of DMH-treated mice resulted in a statistically significant suppression of angiosarcomas and nodular hyperplasia of the liver and colon carcinogenesis. The results of this study also indicate that BBI, included as 0.5% of the diet or less had no adverse effect upon the health of the mice but had the capacity to suppress liver and colon carcinogenesis.
The use of a BBI product was shown to modify the development of lung tumors induced chemically by intraperitoneal (i.p.) injection of 3-methylcholanthrene (MCA) in male strain A mice. Witschi et al., Carcinogenesis 1989, 10, 2275-2277. BBI products have been shown to act as chemopreventative agents to suppress the development of esophageal carcinogenesis induced by i.p. injection of N-nitrosomethylbenzylamine (NMBzA) in male rats. von Hofe et al., Carcinogenesis 1991, 12, 2147-2150. It has also been shown that BBI products effectively reduce the incidence of dimethylhydrazine-induced adenocarcinomas of the colon of mice. Billings et al., Carcinogenesis 1990, 11, 1083-1086.
Various processes have been disclosed for the preparation of different forms of BBI products. Perlmann et al., Methods in Enzymology 1970, 19,860-861, have described an elaborate method for obtaining a BBI product from a defatted soybean extract using an ethanol extraction step.
U.S. Pat. No. 4,793,996 (Kennedy et al.) discloses a process comprising treating soybeans with acetone, followed by ethanol extraction and acetone precipitation for obtaining BBI. The soybeans may be defatted prior to acetone treatment. In addition, BBI may be further purified by conventional techniques. Kennedy et al. discovered that in the conventional process for preparing BBI from soybeans, a factor remained which adversely affected the ability of BBI to inhibit the malignant transformation of cells. If the factor was removed, the resulting BBI product was capable of inhibiting the malignant transformation of cells. It was found to be possible to remove this factor by treating the soybeans with acetone prior to the ethanol extraction step used by Perlmann et al.
Kennedy et al. teach that it is unnecessary to carry out a procedure requiring complete purification of the extract to the point where the product contains only a single protein, but instead it has been found effective to stop the purification procedure at a point where a crude inhibitor extract is obtained. This crude extract (i.e., concentrate) is itself edible and can be used as an inhibitor of malignant transformation of cells, for example by oral ingestion. Kennedy et al. disclose a process for preparing a crude soybean extract containing an inhibitor of malignant cell transformation which comprises defatting soybeans and extracting said inhibitor from said defatted soybeans; the improvement comprises defatting said soybeans by bringing them into contact with at least an equal weight of acetone and, thus, producing a crude inhibitor extract having greatly increased effectiveness.
Kennedy et al., in U.S. Pat. No. 5,217,717 entitled "Methods of Making Soybean Bowman-Birk Inhibitor Concentrate and Use of Same As a Human Cancer Preventative and Therapy", which is incorporated herein in its entirety, describes the methods for producing novel BBI concentrate products. Those BBI concentrate products are employed by the methods of the present invention. The process described to produce those BBI products was found to be economically superior due to the avoidance of an aqueous alcohol extraction step and the use, in certain embodiments, of ultrafiltration as a separation process step.